Lignan complexes

ABSTRACT

An inclusion complex of a lignan or lignan ester with a cyclodextrin. Also disclosed are food products, dietary supplements and pharmaceutical compositions containing the complex.

FIELD OF THE INVENTION

This invention relates to cyclodextrin complexes of lignans or lignanesters, and to the use of such complexes in various food compositions,dietary supplement products or pharmaceuticals.

BACKGROUND OF THE INVENTION

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference.

Cyclodextrins and their Use:

Cyclodextrins (CDs) are a group of cyclic oligosaccharides which havebeen shown to improve pharmaceutical properties of lipophilic drugs byforming inclusion complexes (Frömming K-H, Szejtli J, Cyclodextrins inpharmacy, Kluwer Academic Publishers, Dordrecht, 1994). Cyclodextrinsare cone-shaped molecules with two openings. The cavity of the moleculeis hydrophobic while the surface of the molecule is hydrophilic. Aninclusion complex is formed when the lipophilic guest molecule, or partof it, enters into the apolar cavity of the cyclodextrin. Inclusioncomplex formation is mainly based on hydrophobic interactions betweendrug and cyclodextrin, and no covalent bonds are formed during thecomplexation.

Cyclodextrins are either natural cyclodextrins or derivatives thereof(Thompson D: Cyclodextrins-enabling excipients: their present and futureuse in pharmaceuticals. Crit. Rev. Ther. Drug Carrier Syst. 14: 1-104,1997). Natural cyclodextrins are enzymatic degradation products ofstarch, formed from six (α-cyclodextrin or α-CD), seven (β-cyclodextrinor β-CD) or eight (γ-cyclodextrin or γ-CD) glucopyranose units. Modifiedcyclodextrins, such as methyl-, hydroxyalkyl-, and sulfoalkyletherderivatives of natural cyclodextrins, have been developed to increasethe aqueous solubility and pharmaceutical usefulness of naturalcyclodextrins. So far, the most commonly studied cyclodextrin derivativein drug development is hydroxypropyl-β-cyclodextrin (HP-β-CD).

Cyclodextrins have traditionally been used to increase the aqueoussolubility and chemical/physical stability of lipophilic drugs (LoftssonT, Brewster M E: Pharmaceutical applications of cyclodextrins. 1. drugsolubilization and stabilization. J. Pharm. Sci. 85: 1017-1025, 1996).However, the complexation of a drug with cyclodextrins may also increaseits bioavailability or decrease side-effects (Rajewski R A, Stella V J:Pharmaceutical applications of cyclodextrins 2. in vivo drug delivery.J. Pharm. Sci. 85: 1142-1169, 1996). In addition (as with food andcosmetics preparations), cyclodextrins have also been studied in drugformulations to mask the unpleasant taste or odour of drugs (Frömmingand Szejtli 1994). Until now, the utilization of cyclodextrins has beenlimited to relative small molecules, but cyclodextrins are also usefulwith macromolecules (e.g., proteins and peptides) which will extendtheir utilization in the future (Irie T, Uekama K: Cyclodextrins inpeptide and protein delivery. Adv. Drug Del. Rev. 36: 101-123, 1999).

A problem with natural β-CD is that it causes nephrotoxicity afterparenteral administration (Irie T, Uekama K: Pharmaceutical applicationsof cyclodextrins. III. Toxicological issues and safety evaluation. J.Pharm. Sci. 86:147-162, 1997). However, in oral administration β-CD doesnot show any toxicity due to its minor absorption from thegastrointestinal tract. Similarly, the other natural cyclodextrins andderivatives thereof do not absorb from the gastrointestinal tract due tothe bulky and hydrophilic character of cyclodextrin molecules. In thegastrointestinal tract, cyclodextrins (except for natural γ-CD) areremarkably resistant to the usual starch hydrolysing enzymes. Thecyclodextrins cannot be hydrolyzed by β-amylase and they are hydrolysedby α-amylase at a very low rate. The fundamental physiologicaldifference between cyclodextrins and starch is that the metabolism ofcyclodextrins takes place in the colon while starch is metabolized inthe small intestine. The metabolites of cyclodextrins (maltose, glucose,acyclic maltodextrins) are rapidly metabolized further and finallyexcreted as CO₂ and H₂O. In general, introduction of substituents on thehydroxyl groups slows down enzymatic hydrolysis of the cyclodextrin bylowering its enzyme affinity.

Lignans:

Lignans are phenolic compounds widely distributed in plants. They can befound in different parts (roots, leafs, stem, seeds, fruits) but mainlyin small amounts. In many sources (seeds, fruits), lignans are found asglycosidic conjugates associated with fiber component of plants. Themost common dietary source of mammalian lignan precursors are unrefinedgrain products. The highest concentrations in edible plants have beenfound in flaxseed, followed by unrefined grain products, particularlyrye.

Considerable amounts of lignans are also found in coniferous trees. Thetype of lignans differs among different tree species and the amounts oflignans varies between different parts of the tree. The typical lignansin heartwood of Norway spruce (Picea abies) are hydroxymatairesinol(HMR), alpha-conidendrin, alpha-conidendric acid, matairesinol,isolariciresinol, secoisolariciresinol, liovil, picearesinol,lariciresinol and pinoresino (Ekman R: Distribution of lignans in Norwayspruce. Acta Academiae Aboensis, Ser B, 39:1-6, 1979). The far mostabundant single component of lignans in spruce is HMR, about 60 percentof total lignans, which occurs mainly in non-glycosylated form.

Plant lignans such as HMR, matairesinol, lariciresinol andsecoisolariciresinol, are converted by gut microflora to mammalianlignans, enterolactone or enterodiol. The mammalian lignans can also bemanufactured synthetically (M B Groen and J Leemhius, TetrahedronLetters 21, 5043, 1980).

Lignans are known to possess beneficial effects on human health. Thehealth benefits obtained with lignan rich diet are, for example,decreased risk for various cancers and cardiovascular diseases(Adlercreutz (1998) Phytoestrogens and human health, In: Reproductiveand Developmental Toxicology (edited by Korach, K.). pp. 299-371, Marcel& Dekker, NY.).

Lignans, such as HMR, WO 00/59946, have also been reported to inhibitlipid peroxidation and LDL oxidation and thus be useful as antioxidants.

Also lignans other than HMR have powerful antioxidant andanti-inflammatory potential. The antioxidant action involves all themajor free radicals such as superoxide anions and peroxyl radicals (KPrasad: Antioxidant activity of secoisolariciresinol diglucoside-derivedmetabolites, secoisolariciresinol, enterodiol and enterolactone. Int JAngiology 9:220-225 (2000)).

While literature discloses several different chemical substances thatcan be complexed with different cyclodextrins, the cyclodextrincomplexes of lignans or their derivatives have not been reported so far.

OBJECTS AND SUMMARY OF THE INVENTION

There is a great need to provide novel improved formulations containinglignans or derivatives thereof for use as various kinds of foodproducts, dietary supplements or pharmaceutical use in whichformulations the solubility, bioavailability and stability of the activecompound is satisfactory. Furthermore, masking of possible unpleasanttaste or odour of the active compounds is also important.

Thus, according to one aspect, this invention concerns an inclusioncomplex of a lignan or lignan ester with a cyclodextrin, wherein thelignan or lignan ester is a compound of formula (I)

wherein L is a lignan skeleton which optionally includes a bridgeforming a ring with one of the phenyl groups in the formulae (I) or(II); R₁ is H or methoxy,

-   and R is H, methyl, R′—CO— or R′—SO₂—, wherein-   R′ is a C₁ to C₂₂ alkyl, alkenyl, arylalkyl, aralkenyl, or an    aromatic group, and-   R′ is unsubstituted or substituted with one or more hydroxy groups    and/or one or more carboxyl groups, an oxo group or an amino group,-   or a geometric isomer or a stereoisomer thereof, provided that R is    methyl only in a single R—O— substituent in a compound of    formula (I) where L is a skeleton of the lignan arctigenin.

According to another aspect, this invention concerns a food productcomprising said inclusion complex and a foodstuff.

According to a third aspect, the invention concerns a dietary supplementcomposition comprising said inclusion complex and an acceptable carrier.

According to a fourth aspect, the invention concerns a pharmaceuticalcomposition comprising said inclusion complex and an acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of B-type phase-solubility diagram, where theconcentration of the active compound is shown on the y-axis andcyclodextrin concentration on the x-axis.

FIG. 2 shows a phase-solubility diagram of hydroxymatairesinol (HMR)with γ-cyclodextrin (γ-CD)

FIG. 3 shows a phase-solubility diagram of hydroxymatairesinol diacetate(HMRdiAc) with γ-cyclodextrin

FIG. 4 shows a phase-solubility diagram of matairesinol (MR) withγ-cyclodextrin

FIG. 5 shows a phase-solubility diagram of matairesinol dibutyrate(MRdiBu) with γ-cyclodextrin

FIG. 6 shows a phase-solubility diagram of secoisolraiciresinol (SECO)with γ-cyclodextrin

FIG. 7 shows a phase-solubility diagram of hydroxymatairesinol (HMR)with hydroxypropyl-α-cyclodextrin (HP-β-CD)

FIG. 8 shows the degradation of hydroxymatairesinol as function of timein the presence (squares) or absence (triangles) of 2% γ-cyclodextrin.

DETAILED DESCRIPTION OF THE INVENTION

Preferred Cyclodextrins:

Although any natural cyclodextrin or derivative thereof could beemployed in this invention, natural α-, β- or γ-cyclodextrins arepreferred. Particularly preferred is γ-cyclodextrin. Preferredderivatives are methyl-, hydroxyalkyl- and sulfoalkylether derivativesof natural cyclodextrins. An especially preferred cyclodextrinderivative is hydroxypropyl-β-cyclodextrin.

Preferred Lignans and Lignan Esters:

As can be seen in Scheme 1, lignans bear typically two phenyl groups,which in turn are substituted with at least a hydroxy group. Anexception is the lignan arctigenin in which one of the phenolic hydroxylgroups is replaced by methoxy. Most of the lignans of formula (I) havedisubstituted phenyl groups, i.e. R₁ is H. An exception is the ryelignan syringaresinol in which R₁ is methoxy. The lignan skeleton L inthe formulae (I) and (II) stands for the part of the lignan moleculebearing such phenyl groups. In certain lignans such as isolariciresinoland conidendrin, the skeleton L includes a bridge which forms a ringwith one of the phenyl groups in the formulae. As further can be seen,many of the lignans have also one or more hydroxy groups in the skeletonL.

Preferred lignans are lignans according to formula (I) which arehydroxymatairesinol, matairesinol, lariciresinol, secoisolariciresinol,isolariciresinol, oxomatairesinol, alpha-conidendrin, pinoresinol,liovil, picearesinol, arctigenin, syringaresinol or nortrachelogenin, orlignans of formula (II), which are enterolactone or enterodiol.

Especially preferred lignans are hydroxymatairesinol, matairesinol,lariciresinol, secoisolariciresinol and isolariciresinol and theirgeometric isomers and stereoisomers.

“Esters” of lignans shall mean either phenolic esters (where the hydroxygroups in the phenol are esterified) or esters where hydroxysubstituents in the lignan skeleton are esterified. Many esters of thelatter kind are disclosed in the art. Certain phenolic lignan esters arealso known in the art, namely the dibenzoate and the p-nitrodibenzoateof matairesinol; enterolactone diacetate; monoacetate, triacetate,p-hydroxymonobenzoate, and p-hydroxy-m-methoxymonobenzoate ofhydroxymatairesionol; and tetraacetate and tetrabenzoate ofsecoisolariciresinol. Other phenolic diesters of lignans defined byformulas (I) or (II) have recently been disclosed in a patentapplication.

The ester is preferably a phenolic ester, in particular a phenolicdiester.

Preferable diphenolic lignan esters are, for example, esters of mono- ordicarboxylic fatty acids, hydroxy acids and sulfonic acids. As examplesof suitable dicarboxylic acid lignan esters can be mentioned succinates,glutarates, and malonic acid esters. Lactic acid esters are examples ofesters with hydroxysubstituted acids. Tartaric acid and citric acidesters are examples of esters of acids with several carboxylic groupsand one or more hydroxy groups.

Preparation of the Inclusion Complex:

The cyclodextrin inclusion complex of the lignans or lignan esters arepreferably prepared by adding the compound to the cyclodextrin in anacetate buffer at pH 5. The complex formed can be precipitated andisolated.

However, the solid inclusion complex of lignan and cyclodextrin can alsobe prepared simply by freeze-drying or spray-drying the solution. Inaddition, methods such as kneading, grinding, neutralization andso-called slurry methods have been used to prepare solid inclusioncomplexes.

The inclusion complex according to this invention can be provided in theform of a pharmaceutical preparation, dietary supplement, or a foodproduct.

The pharmaceutical preparation is preferably an oral formulation. Therequired amount of the active compound or mixture of compounds will varywith the compound and the particular condition to be prevented. Atypical dose ranges from about 1 to about 2000 mg (calculated as lignan)per day and adult person, preferably 10 to 600 mg per day and adultperson. Typical dosage forms include, but are not limited to, oraldosage forms such as powders, granules, capsules, tablets, caplets,lozenges, liquids, elixirs, emulsions and suspensions. All such dosageforms may include conventional carriers, diluents, excipients, bindersand additives known to those skilled in the medicinal and pharmaceuticalarts.

The carriers typically employed for the pharmaceutical composition ordietary supplement composition may be solid or liquid. Thus, forexample, solid carriers include polysaccarides such as lactose, sucrose,gelatin, agar, while liquid carriers include aqueous solutions of salts,polysaccarides, complexing agents, surfactants, syrups, vegetable oilssuch as peanut oil or olive oil, and certain alcohols. However, anyacceptable solid or liquid carrier can be used in the pharmaceuticalpreparation or other dietary or nutrition formula to be administeredaccording to this invention.

A typical food product, suitable for use in the methods according tothis invention, is especially a functional food, a nutritionalsupplement, a nutrient, a pharmafood, a nutraceutical, a clinicalnutritional product, a health food, a designer food or any food product.The term food product shall also be understood to cover groceries andfoodstuffs such as flour, other ingredients, certain liquids etc. Asuitable concentration of the active compound in the food product is,for example, 1 to 1000 mg of active compound per 100 g of food product,preferably about 10 to 100 mg of active compound per 100 g of foodproduct.

The invention will be illuminated by the following non-restrictiveExperimental Section.

EXPERIMENTAL SECTION

Materials and Methods

Chemicals

The lignans and lignan esters (hydroxymatairesinol (HMR), matairesinol(MR), hydroxymatairesinol diacetate (HMRdiAc), matairesinol dibutyrate(MRdiBu) and secoisolariciresinol (SECO)) were received from HormosNutraceutical Ltd. and natural γ-CD and HP-β-CD was purchased fromWacker-Chemie GmbH (Burghausen, Germany). All other chemicals used wereof analytical grade.

Apparatus

The samples from the solubility and stability studies were analysed byusing the HPLC system which consist of the UV-detector (L-7400),interface module (D-7000), pump (L-7100) and autosampler (L-7250, MerckHitachi, Japan). Purospher® reversed phase column (RP-18e, 5 μm, 125×4mm) was used in all chromatographic separations.

Solubility Studies

The complexation of lignans and lignan esters with γ-CD was studied byusing the phase-solubility method of Higuchi and Connors (Higuchi T,Connors K. A. Phase-solubility techniques. Adv. Anal. Chem. Instr. 4:117-212, 1965). An excess amount of lignan or lignan ester was added toacetate buffer (0.16 M; pH 5.0; ionic strength 0.5) containing variousconcentrations of γ-CD (0-10%). The suspensions were shaken in the dark(25° C.) for 24 hour and the pH of the suspensions were monitored duringthe equilibration. The pH of suspensions was adjusted to 5.0 with HCl orNaOH if necessary. After equilibration, the suspensions were filteredthrough 0.45 μm membrane filters and analysed by HPLC.

The phase solubility studies with HMR was also performed with HP-β-CD(0-10%). In the case of HP-β-CD the equilibration time was 72 hours.

Stability Studies

The chemical stability of HMR was studied in acetate buffer (0.16 M; pH5.0; ionic strength 0.5) in the presence and absence of 2% γ-CD at 30°C. All the solutions were prepared by dissolving 1.5-2.0 mg of HMR into20 ml of the solutions mentioned above, and the concentration of theremaining HMR was determined at appropriate intervals by HPLC. Thepseudo-first order rate constant for overall degradation of HMR wasdetermined from the slopes of the linear semilogarithmic plots ofremaining HMR versus time. The results of the stability studies werecalculated as an average of three determinations.

Results

FIG. 1 shows an example of the B-type phase-solubility diagram. InB-type phase-solubility diagram the concentration of the activecompound, e.g. the complexed drug, first increases with increasingcyclodextrin concentration due to complexation of the active compoundwith the cyclodextrin molecules. However, after initial improvement incompound solubility, the maximum solubility of the complex is achievedand no further improvement is reached with increasing cyclodextrinconcentration (highest part of the diagram). At a certain cyclodextrinconcentration, the solubility of the compound begins to decrease in theB-type phase-solubility diagram, because at high cyclodextrinconcentrations the compound forms lower solubility complexes withcyclodextrins. The B-type phase-solubility behaviour is typical fornatural cyclodextrins and has been described earlier e.g. with steroidhormones (Uekama K, Fujinaga T, Hirayama F, Otagiri M, Yamasaki M.Inclusion complexation of steroid hormones with cyclodextrins in waterand in solid phase. Int. J. Pharm. 10: 1-15, 1982).

Solubility Studies

Table 1 shows the effect of γ-CD on the aqueous solubility of theselected lignans and lignan esters at pH 5.0. The same data are alsoshown in FIGS. 2-6 showing the phase-solubility data in graphical form.FIGS. 2-6 show that all the lignans and lignan esters studied formB-type phase solubility diagram with γ-CD. TABLE 1 The effect of γ-CD onthe aqueous solubility of lignans (0.16 M acetate buffer; pH 5.0; μ =0.5, 25° C.; equilibration time 24 hours) HMR HMRdiAc MR MRdiBu SECOγ-CD (g/100 ml) (mg/ml) (mg/ml) (mg/ml) (μg/ml) (mg/ml) 0 6.70 0.75 0.49—* 0.70 1 8.85 1.46 2.27 —* 1.74 2 10.80 2.07 1.69 0.87 2.02 5 5.50 0.941.47 1.21 1.78 10 2.21 0.16 0.41 0.84 0.39*= concentration is under the detection limit of the HPLC methodemployed.

The complexation of HMR was also studied with HP-β-CD which is the mostcommonly used cyclodextrin derivative in drug development at present.The results (Table 2) show that HP-β-CD forms an inclusion complex withHMR and increases aqueous solubility of HMR. The same data are alsoshown in FIG. 7. TABLE 2 The effect of HP-β-CD on aqueous solubility ofHMR (0.16 M acetate buffer; pH 5.0; μ = 0.5; 25° C.; equilibration time72 hours) HP-β-CD concentration Aqueous solubility (g/100 ml) of HMR(mg/ml) 0 14.56 1 14.90 2 14.94 5 20.16 10 26.83Stability Studies

The overall degradation of HMR followed first-order kinetics in thepresence and absence of 2% γ-CD at pH 5.0 (FIG. 8). Table 3 shows thefirst-order rate constants, half-lives (t_(1/2)) and shelf-lives(t_(90%)) for the chemical degradation of HMR.

The stability studies showed that 2% of γ-CD increases the chemicalstability of HMR about 12-fold at pH 5.0. TABLE 3 First-order rateconstants (k_(obs)), half-lives (t_(1/2)) and shelf-lives (t_(90%)) forchemical degradation of HM-3000 at pH 5.0 (30° C.). Vehicle k_(obs)(h⁻¹) t_(1/2) (h) t_(90%)(h) 0.16 M Acetate buffer (pH 5.0) Without γ-CD6.91 × 10⁻⁴ 1003 152 With 2% γ-CD 5.71 × 10⁻⁵ 12134 1845

CONCLUSIONS

The results show that lignans and lignan esters form complexes withnatural γ-cyclodextrin. With all the lignans and lignan esters studied,γ-CD complexation increased the aqueous solubility of the compounds atlow γ-CD concentrations. However, at high γ-CD concentrations lignansand lignan esters form higher order complexes with natural γ-CD whichresults in decreased aqueous solubility (B-type phase-solubilitybehaviour). The main advantage of the B-type phase-solubility behaviouris the simple and effective preparation of the pure inclusion complexesby precipitation. Usually CD-containing products are the mixtures ofnon-complexed molecules of the active agent, complexed molecules of theactive agent, and “empty” CD molecules. However, the B-type phasesolubility behaviour allows the preparation of pure cyclodextrincomplexes of the active agent, i.e. no free cyclodextrin molecules andmolecules of the active agent are present in the product.

The present study also shows that the complexation of HMR with γ-CDsignificantly increases the aqueous stability of HMR. The present studywas carried out at 2% γ-CD concentration where HMR has the bestsolubility and the stoichiometry of the complex is most probably 1:1.Thus, it might be possible to improve the aqueous stability of HMRfurther by increasing the CD concentration which also changes thestoichiometry of the complex.

In addition, the present study shows that HP-β-CD can be used to improvethe aqueous solubility of HMR.

The present study shows that the solubility of HMR without CDs is highlydependent on the equilibration time. Thus, it is important to point outthat one of the major benefits of cyclodextrine complexation of lignansor lignan esters may also be the significant improvement the dissolutionrate of the compounds, because cyclodextrins have been shown to increasethe dissolution rate of lipophilic compounds in various applications.

It will be appreciated that the methods of the present invention can beincorporated in the form of a variety of embodiments, only a few ofwhich are disclosed herein. It will be apparent for the expert skilledin the field that other embodiments exist and do not depart from thespirit of the invention. Thus, the described embodiments areillustrative and should not be construed as restrictive.

1. An inclusion complex of a lignan or lignan ester with a cyclodextrin,wherein the lignan or lignan ester is a compound of formula (I)

wherein L is a lignan skeleton, which optionally includes a bridgeforming a ring with one of the phenyl groups in the formulae; R₁ is H ormethoxy, and R is H, methyl, R′—CO— or R′—SO₂—, wherein R′ is a C₁ toC₂₂ alkyl, alkenyl, arylalkyl, aralkenyl, or an aromatic group, and R′is unsubstituted or substituted with one or more hydroxy groups and/orone or more carboxyl groups, an oxo group or an amino group, or ageometric isomer or a stereoisomer thereof, provided that R is methylonly in a single R—O— substituent in a compound of formula (I) where Lis a skeleton of the lignan arctigenin.
 2. The complex according toclaim 1 wherein the cyclodextrin is α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin or a derivative thereof.
 3. The complex according toclaim 2 wherein the cyclodextrin is natural cyclodextrin.
 4. The complexaccording to claim 2 wherein the cyclodextrin is γ-cyclodextrin orhydroxypropyl-β-cyclodextrin.
 5. The complex according to claim 1,wherein L in compound (I) is a lignan skeleton of any of the lignansselected from the group consisting of hydroxymatairesinol, matairesinol,lariciresinol, secoisolariciresinol, isolariciresinol, oxomatairesinol,alpha-conidendrin, pinoresinol, liovil, picearesinol, arctigenin,syringaresinol and nortrachelogenin.
 6. The complex according to claim1, wherein L in compound (II) is a lignan skeleton of enterolactone orenterodiol.
 7. The complex according to claim 1, wherein both of thegroups R—O— in the compound (I) or (II) are hydroxy groups.
 8. Thecomplex according to claim 1, wherein both of the groups R—O— in thecompound (I) or (II) are ester groups, where R is R′—CO or R′—SO₂ and R′is a C₁ to C₂₂ alkyl, alkenyl, arylalkyl, aralkenyl, or an aromaticgroup, and R′ is unsubstituted or substituted with one or more hydroxygroups and/or one or more carboxyl groups, an oxo group or an aminogroup or a geometric isomer or a stereoisomer thereof.
 9. A food productcomprising a complex according to claim 1 and a foodstuff.
 10. The foodproduct according to claim 9, which is a functional food, a nutritionalsupplement, a nutrient, a pharmafood, a nutraceutical, a clinicalnutrition product, a health food or a designer food.
 11. A dietarysupplement composition comprising a complex according to claim 1 and anacceptable carrier.
 12. A pharmaceutical composition comprising acomplex according to claim 1 and an acceptable carrier.